This research project involves the genetic and biochemical characterization of a collection of temperature-sensitive mutants of Escherichia coli, isolated by localized mutagenesis of the bacterial chromosome. Many of these are defective in protein synthesis, both in vivo and in cell free assays. Several have shown alterations in single ribosomal proteins. Others are defective in the assembly of ribosomal subunits at the restrictive temperature (44 degrees C). Our research effort includes the isolation and characterization of the altered ribosomal proteins from these mutants. We are also conducting genetic mapping studies to determine the chromosomal location of the mutations. Assays are being conducted to determine the precise defects in ribosome function accompanying the ribosomal protein changes. We are also attempting an identification of the ribosomal RNA and protein species present in the accumulated precursor particles. Finally, a novel mutant in our collection displays an osmotic remedial conditional temperature-sensitivity. Changes in a protein located in the periplasmic space can be observed to correlate with changes in the temperature-sensitive phenotype. We are presently examining this mutant strain for alterations in membrane permeability.